Methods of treating porphyria

ABSTRACT

A method of treating porphyria in a patient is provided comprising knocking down or reducing expression or activity of β-catenin in the patient, e.g., in the liver of a patient, to an extent effective to treat porphyria in a patient.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/697,548 filed Jul. 13, 2018, which is incorporatedherein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos.DK062277; DK100287 and DK103775, awarded by the National Institutes ofHealth. The government has certain rights in the invention.

REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The content of the ASCII text file of the sequence listing named“1903391_ST25” which is 36,729 bytes in size was created on Jun. 18,2019 and electronically submitted via EFS-Web herewith the applicationis incorporated herein by reference in its entirety.

Provided herein are methods of treating porphyria.

Porphyrins are tetrapyrrole compounds and heme precursors, with hemebeing a co-factor for functionally diverse hemoproteins such ascytochrome-P450, hemoglobin, and peroxidases. Heme biosynthesis startsin mitochondria, where glycine and succinyl Co-A are combined to formthe first committed metabolite of the pathway, δ-aminolevulinic acid(ALA), catalyzed by ALA-synthase (ALA-S). ALA leaves the mitochondria,and is sequentially converted in the cytosol to porphobilinogen,hydroxymethylbilane, uroporphyrin (Uro) and then to coproporphyin(Copro), which re-enters mitochondria. In the penultimate step,protoporphyrin-IX is generated, which is metallated by ferrochelatase toform the iron containing heme.

3,5-Diethoxycarbonyl-1,4-dihydrocollidine (DDC), a porphyrinogeniccompound, has been widely used to induce hepatic porphyria andMallory-Denk bodies in mouse models. DDC perturbs porphyrin biosynthesisat multiple steps. DDC N-methylates heme residues of some hepaticcytochrome-P450 enzymes, which subsequently demetallates and releasesthe tetrapyrrole moiety in the form of N-methyl protoporphyrin IX.N-methyl protoporphyrin IX is a potent inhibitor of ferrochelatase andblocks conversion of protoporphyrin-IX to heme. Thus, hepatic NMPaccumulation causes buildup of tetrapyrrole precursors, includingprotoporphyrin-IX. Additionally, ALA-S is under negative feedbackregulation of heme. Inhibition of ferrochelatase by N-methylprotoporphyrin IX, and the subsequent heme-deficient state, de-repressesALA-S expression, which in turn generates more ALA that feeds forwardinto the heme biosynthetic pathway, leading to accumulation of toxicporphyrins.

DDC has also been used widely to study formation of Mallory-Denk bodies,which are hepatocellular inclusions found in diseases like alcoholic andnon-alcoholic steatohepatitis and metabolic liver diseases. Aggregatesof keratin proteins K8 and K18 constitute a major component ofMallory-Denk bodies as they are early sensors of porphyrin-mediatedliver injury. Protein aggregation perturbs protein clearance, causingproteasomal inhibition and induction of autophagy. Chronic DDC exposurealso results in pericholangitis, ductular reaction, oval cell responseand periductal fibrosis, similar to that seen in primary sclerosingcholangitis patients, owing to crystallized porphyrin obstructing smallbile ducts.

SUMMARY OF THE INVENTION

A method of treating porphyria in a patient is provided. The methodcomprises: decreasing expression or activity of β-catenin in thepatient, e.g., in the patient's liver, and optionally targeting deliveryto the patient's liver, thereby decreasing porphyrin production and/oraccumulation, or reducing one or more symptoms of porphyria, such asliver injury in the patient. Related reagents and uses for the reagentsalso are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1: An exemplary, but non-limiting sequence for β-catenin (SEQ IDNO: 1), from Homo sapiens catenin beta 1 (CTNNB1), mRNA transcriptvariant 1.

FIG. 2 provides structures of ICG001 and PRI 724.

FIGS. 3A-3C provide structures of exemplary PORCN inhibitors.

FIGS. 4A-4F: Alb-cre β-catenin KO have lesser injury than WT after 2weeks of DDC. (FIG. 4A) Treatment regimen for WT1 and Alb-cre β-cateninKO (KO1) on DDC. (FIG. 4B) Gross liver specimens from WT1 and KO1 miceon 2 week DDC demonstrate that WT1 livers become enlarged and turn darkbrown in color, while the KO1 livers are smaller and lighter in color(right panel). Liver weight to body weight ratios are lower in KO1compared to WT1 after DDC (left panel). (FIG. 4C) Both biliary andhepatocyte injury are decreased in KO1 after DDC, as assessed by ALP andALT. (FIG. 4D) Glutamine synthetase IHC confirms loss of β-catenin inKO1. (FIG. 4E) H&E shows lesser porphyrin accumulation in KO1 comparedto WT1 after DDC. (FIG. 4F) Sirius red staining shows equivalentfibrosis in KO1 after DDC compared to WT1. n=3 for WT1 and KO1 baseline,n=5 WT1+DDC, n=4 KO1+DDC; *P<0.05, **P<0.01 by Student's t test.

FIGS. 5A-5B: No change in biliary/cholangiocyte markers after β-cateninor Wnt signaling deletion and DDC. (FIG. 5A) WB demonstrates absentβ-catenin and cyclin D1 expression in β-catenin KO (KO1) livers. Yapexpression increases in KO1; however, the inactivated, S127 p-Yap formalso increases as well. (FIG. 5B) WB shows that in LRP5/6 KO (KO2)livers after DDC, β-catenin is still present; however, cyclin D1 issignificantly decreases. Yap and p-Yap are not significantly changedbetween WT2 and KO2.

FIGS. 6A-6C: Proliferation and inflammation are decreased in KO1 liversafter DDC. (FIG. 6A) IHC shows a significant decrease in both cyclin D1and Ki67 staining in KO1 after DDC. (FIG. 6B) Ductular response isequivalent in WT1 and KO1 livers, as assessed by EpCAM and Sox9 IHC.(FIG. 6C) There is lesser inflammation in KO1 livers after DDC, asdemonstrated by decreased CD45, CD68, and neutrophil elastase stainingin KO1 livers. For IHC, n≥3 liver sections from n≥3 animals from bothgroups were analyzed; representative images are shown.

FIGS. 7A-7G: Alb-cre LRP5/6 KO have lesser injury than WT after DDC.(FIG. 7A) Treatment regimen for WT2 and Alb-cre LRP5/6 KO (KO2) on DDC.(FIG. 7B) Gross liver specimens show that WT2 livers are larger anddarker than KO2 livers after DDC. (FIG. 7C) Liver weight to body weightratios are lower in KO2 compared to WT2 after DDC. (FIG. 7D) Biliaryinjury is decreased in KO2 after DDC, as assessed by lower ALP serumlevels. (FIG. 7E) Glutamine synthetase IHC confirms loss of β-catenintranscriptional activation in KO1. (FIG. 7F) H&E shows a modest decreasein porphyrin accumulation in KO2 compared to WT2 after DDC. (FIG. 7G)Sirius red staining shows equivalent fibrosis in KO2 after DDC comparedto WT. n=3 for WT2 and KO2 baseline, n=5 WT2+DDC, n=4 KO2+DDC;***P<0.001; ****P<0.0001 by Student's t test.

FIGS. 8A-8C: Like KO1, KO2 livers have decreased cyclin D1 expressionand hepatic inflammation after DDC. (FIG. 8A) IHC shows a significantdecrease in cyclin D1 in KO2 after DDC; however, hepatocyteproliferation appears to be unimpaired. (FIG. 8B) Ductular response isequivalent in WT2 and KO2 livers, as assessed by EpCAM and Sox9 IHC.(FIG. 8C) Inflammation is deceased in KO2 livers after DDC, as shown bydecreased CD45, F4/80, and neutrophil elastase staining. For IHC, n≥3liver sections from n≥3 animals from both groups were analyzed;representative images are shown.

FIGS. 9A-9F: Exogenous inhibition of β-catenin using DsiRNA results indecreased injury after 4 weeks of DDC. (FIG. 9A) Treatment regimen foradministration of DsiRNAs after DDC. (FIG. 9B) Western blot confirmsdecreased β-catenin protein after DsiRNA treatment. (FIG. 9C) Grossliver specimens demonstrate that control livers become enlarged and darkred after DDC, while β-catenin DsiRNA treated livers are smaller andlighter in color. Liver weight to body weight ratios confirm decrease inβ-catenin DsiRNA-treated livers after DDC. (FIG. 9D) Both biliary andhepatocyte injury are decreased after β-catenin DsiRNA and DDC, asassessed by ALP and ALT. (FIG. 9E) Sirius red staining shows a reductionin fibrosis after DDC and β-catenin DsiRNA treatment compared to controlDsiRNA and DDC. (FIG. 9F) Channel splitting and quantification ofrepresentative Sirius red images demonstrates a significant decrease inpercent area of fibrosis in β-catenin DsiRNA treated livers after DDC.For C, n=3 for control and n=4 for β-catenin siRNA at baseline, n=5control+DDC, n=4 β-catenin+DDC; for D, n=3 for control and n=4 forβ-catenin siRNA at baseline, n=11 control+DDC, n=11 β-catenin+DDC;*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 by Student's t test.

FIGS. 10A-10E: DsiRNA for β-catenin suppresses proliferation andinflammation after DDC. (FIG. 10A) GS IHC confirms loss of β-catenin inhepatocytes after DsiRNA treatment. (FIG. 10B) α-SMA expression isequivalent in control and β-catenin DsiRNA treated livers after DDC.(FIG. 10C) Hepatocyte proliferation is decreased in livers afterβ-catenin DsiRNA treatment, while cholangiocyte proliferation isunaffected. (FIG. 10D) Inflammatory markers CD45 and F4/80 are decreasedafter β-catenin DsiRNA, while neutrophil elastase is increased. (FIG.10E) Sox9-positive cells extend into the parenchyma after β-cateninDsiRNA treatment and DDC. For IHC, n≥3 liver sections from n≥3 animalsfrom both groups were analyzed; representative images are shown.

FIGS. 11A-11B: No change in biliary/cholangiocyte markers afterβ-catenin inhibition and DDC. (FIG. 11A) The ratio of Yap to p-Yap isnot significantly different between control DsiRNA and β-catenin DsiRNAtreated livers after DDC. (FIG. 11B) Jagged1 mRNA expression isincreased after β-catenin DsiRNA treatment and DDC compared to control;however, Notch1 expression is significantly decreased. n=3 mice pergroup analyzed in duplicate; *P<0.05 by Student's t-test.

FIGS. 12A-12B: Protection from DDC in β-catenin DsiRNA treated livers isnot due to NF-κB activation or decreased bile acid accumulation. (FIG.12A) Transcription factor assay shows no significant changes in NF-κBactivity between control and β-catenin DsiRNA livers after DDC. (FIG.12B) Total bile acid accumulation in the liver is equivalent in controland β-catenin DsiRNA treated livers. (FIG. 12A) n=4 mice per groupanalyzed in triplicate; for (FIG. 12B), n=4 control DsiRNA-treated miceand n=6 β-catenin DsiRNA-treated mice.

FIGS. 13A-13D: There is decreased porphyrin accumulation in β-cateninDsiRNA treated mice after DDC compared to DDC alone. (FIG. 13A) H&Eshows fewer porphyrin inclusions (arrows) in livers treated withβ-catenin DsiRNA and DDC compared to controls. (FIG. 13B) Plotting thepercent porphyrin per image using ImageJ Fiji to separate color channelsshows a quantifiable decrease in porphyrin after β-catenin DsiRNAtreatment and DDC. (FIG. 13C) Determination of total liver porphyrins(except heme) by fluorescence assay confirms less porphyrin accumulationin β-catenin DsiRNA-treated livers. The data is expressed as mean witherror bars representing standard error of measurements. n=3 controlDsiRNA, n=3 β-catenin DsiRNA, n=3 Control DsiRNA+DDC, n=4 β-cateninDsiRNA+DDC. P<0.05; ****P<0.0001 by Student's t-test. (FIG. 13D)Measurement of porphyrin intermediates by LC-MS shows decreased PP-IX inlivers treated with β-catenin DsiRNA after DDC compared to those treatedwith control DsiRNA and DDC. n=3 control DsiRNA, n=3 β-catenin DsiRNA,n=4 Control DsiRNA+DDC, n=4 β-catenin DsiRNA+DDC. *P<0.05; ****P<0.0001by Student's t-test.

FIGS. 14A-14D: β-catenin inhibition alters pathways involved in DDCmetabolism. (FIG. 14A) Both Cyp3a1 and Cyp3a11 are increased in KO1 atbaseline compared to WT1. (FIG. 14B) Expression of 2 Cyp3a isoforms,Cyp3a1 and Cyp3a11, are equivalent after β-catenin DsiRNA at baseline(before DDC). (FIG. 14C) Cyp3a1 protein expression is notably increasedin livers treated with β-catenin DsiRNA and DDC compared to DDC alone.(FIG. 14D) CAR mRNA expression is decreased basally after β-cateninDsiRNA administration. After DDC, CAR expression is suppressed incontrols to a similar extent as β-catenin DsiRNA treated livers beforeDDC. n=3 mice per group analyzed in duplicate. For A, *P<0.05; **P<0.01by Student's t-test. For D, *P<0.05 vs. control DsiRNA, #P<0.05 vs.β-catenin DsiRNA, % P<0.05 vs. control+DDC, all by Student's t-test.

FIGS. 15A-15F: Decreased porphyrin accumulation in β-cateninDsiRNA-treated livers is due to alterations in heme biosynthesis pathwayenzymes. (FIG. 15A) WB shows decrease in ALA-S, the rate-limiting enzymein the heme biosynthesis pathway, after β-catenin DsiRNA and DDC. (FIG.15B) Ferrochelatase (FC) protein expression is equivalent in control andβ-catenin DsiRNA livers after DDC. (FIG. 15C) ALA-aminolevulinic aciddehydratase (ALA-D) is decreased in livers of mice treated withβ-catenin DsiRNA and DDC. (FIG. 15D) Expression of ALA-S and ALA-D aredecreased after β-catenin DsiRNA at baseline (before DDC). (FIG. 15E)ALA-S and ALA-D are also significantly decreased in KO1 at baseline.(FIG. 15F) ALA-D is suppressed in KO2 compared to controls at baseline,while ALA-S is insignificantly decreased. n≥3 mice per group analyzed induplicate; *P<0.05; **P<0.01; ***P<0.001 by Student's t-test.

FIG. 16: The ALA-D gene contains a TCF4 binding site. ChIP-seq mapacross a 50,000 bp region of the ALA-D gene showing binding sites forRXRα, FXR, TCF4, HNF4, as well as H4K5Ac (transcriptionally activechromatin) and H3K4Me3 (active promoter regions) relative to thetranscription start site. TCF4 (red peak) binds to an intron downstreamof the ALA-D promoter. This binding peak coincides with strong peaks forother nuclear receptors such as CAR, RXRα, FXR, and HNF4.

FIGS. 17A-17C: Loss of β-catenin protects mice from DDC-mediateddepletion of heme. Total liver homogenates were separated onnon-reducing non-denaturing PAGE (8% resolving gel: FIG. 17A (panel Aand panel C); 15% resolving gel: FIG. 17A (panel B and panel D), andexposed to film for the indicated times. Ponceau staining of the PVDFmembranes are shown as a loading control, and show equal protein loadingamong different samples. For FIG. 17A (panels A, C), arrowheadsrepresent bottom of the well, stacking-resolving gel junction, and endof the gel, respectively. For FIG. 17 (panels B and D), arrowheadsrepresent the stacking-resolving gel junction. (FIG. 17B) Measurement oftotal heme content shows persistence of heme in livers treated withβ-catenin DsiRNA after DDC compared to those treated with control DsiRNAand DDC. (FIG. 17C) HO-1 mRNA expression is suppressed in the absence ofβ-catenin before and after DDC compared to controls. For FIG. 17B, n=3mice per group; % P<0.05 vs. control+DDC by Student's t-test. For FIG.17C, n=3 mice per group analyzed in duplicate; *P<0.05 vs. controlDsiRNA, #P<0.05 vs. β-catenin DsiRNA, % P<0.05 vs. control+DDC, all byStudent's t-test.

FIG. 18: Loss of β-catenin protects from DDC-mediated destruction ofheme-containing proteins. FIG. 18 (panels A, B, C, and D) Total liverhomogenates were separated on non-reducing denaturing PAGE, and hemecontent assayed as detailed in the Methods section. Ponceau staining ofthe PVDF membranes are shown as a loading control, and show equalprotein loading among different samples.

FIG. 19: β-catenin DsiRNA decreases total porphyrin and alters proteinaggregation after DDC. (Top two panels) SDS-PAGE stained with CoomassieBlue highlights changes in protein banding pattern (Arrows with romannumerals i-v) as a function of DDC in control vs. β-cateninDsiRNA-treated livers. Porphyrin fluorescence normalized to mg ofprotein (top of gel) shows a significant decrease in porphyrinaccumulation after β-catenin DsiRNA treatment and DDC compared tocontrol DsiRNA after DDC. (Bottom panel) DDC feeding also led to markedinduction of GST-mu, visible by Coomassie staining and also validated byimmunoblotting. *P<0.05 by Student's t test.

FIGS. 20A-20C: β-catenin inhibition prevents DDC-mediated nuclear IFprotein damage. FIG. 20A (panel A) Cytosolic K8 protein expression (inboth monomer and HMW aggregate form) is increased in both control andβ-catenin DsiRNA treated livers after DDC. FIG. 20A (panel B) Monomericand aggregate forms of cytosolic K18 is also increased after DDC. FIG.20A (panel C) Loss in monomeric nuclear lamin A/C after DDC wasprevented in β-catenin DsiRNA treated livers. FIG. 20A (panel D)β-catenin DsiRNA treated livers also showed persistence of the momomericform of nuclear lamin B1 after DDC. For FIG. 20A (panels A, B, C, andD), monomer (arrowhead, ‘mono’) appears after a short chemiluminescenceexposure, while HMW aggregates in upper panel are after a longerexposure. (FIG. 20B) Relative change (with respect to control andβ-catenin DsiRNA at baseline) in monomer band intensity after DDCfeeding. (FIG. 20C) Quantification of HMW aggregates after DDC-feedingin control vs. β-catenin DsiRNA treated mice. The area of the blotquantified is shown in solid lined box. *P<0.05.

FIGS. 21A-21F: β-catenin ameliorates DDC-associated perturbation ofprotein clearance and autophagy pathways. (FIG. 21A) WB for IRE1α, anindicator of ER stress and the UPR, shows decreased aggregates and anincrease in the monomeric form after β-catenin DsiRNA and DDC comparedto controls. (FIG. 21B) CHOP, another ER stress marker, is alsoincreased in β-catenin DsiRNA-treated livers after DDC compared to DDCalone. (FIG. 21C) WB for p62, a ubiquitin/proteasomal marker, showspresence of the monomeric form in β-catenin DsiRNA treated liversdespite formation of HMW aggregates due to DDC. (FIG. 21D) WB for Ubshows a similar extent of proteasomal inhibition in both control andβ-catenin DsiRNA-treated mice after DDC. Dotted box indicates increasedaccumulation of HMW ubiquitinated proteins after DDC. (FIG. 21E) WB ofLC3B proteins shows decrease in the amount of the LC3B I form after DDC,while HMW aggregates increase. Top panel shows HMW aggregate, and bottompanel shows LC3B I and II bands. (FIG. 21F) Quantification of bandintensity from immunoblot shown in FIG. 21E demonstrates increasedautophagy in controls after DDC but not in β-catenin DsiRNA treatedlivers (right panel). LC3B aggregates are also decreased in β-cateninDsiRNA treated livers (left panel). *P<0.05.

FIGS. 22A-22B: Model showing the interaction of DDC with hemebiosynthetic pathway, and its modulation by β-catenin. (FIG. 22A) Innormal liver, DDC N-methylates heme to form NMP, which inhibits FC,causing heme depletion and accumulation of porphyrins such as Uro,Copro, and PP-IX. β-catenin regulates the expression of several genes,such as ALA-D and HO-1, that are involved in this process. The netresult is exacerbation of injury when β-catenin is present. (FIG. 22B)In the absence of β-catenin, ALA-D expression decreases, resulting inless PP-IX accumulation and less heme depletion. Loss of β-catenin alsoreduces HO-1 expression, which further prevents heme depletion. Thedecrease in porphyrin intermediates and maintenance of cellular heme inturn reduces ALA-S expression. Purple arrows, effects mediated by DDC;green arrows, effects mediated by β-catenin.

FIGS. 23A-23C: β-Catenin transcriptional inhibitor Wnt-C59 reducesinjury after DCC. (FIG. 23A) Both biliary and hepatic injury aredecreased after DCC in Wnt-C59-treated livers. (FIG. 23B) Wnt-C59suppresses transcriptional activation of Wnt/β-catenin target gene GS.(FIG. 23C) H&E shows less porphyrin in Wnt-C59-treated livers comparedto controls after DCC (100×).

FIG. 24: β-Catenin inhibition reduces basal hepatic ALA-S and ALA-D mRNAlevels in WT1/WT2 mice.

FIGS. 25A-25B: β-Catenin inhibition ameliorates DDC-associated proteinaggregation and promotes autophagy. (FIG. 25A) SDS-PAGE stained withCoomassie Blue highlights changes in protein banding pattern (arrowswith roman numerals i-v) as a function of DDC in control vs. β-cateninDsiRNA-treated livers. (FIG. 25B) WB shows decrease in the amount of theLC3BI form after β-catenin DsiRNA, while LC3BII increases, indicatingincreased autophagy. LC3B aggregates are also decreased in β-cateninDsiRNA+DDC.

FIG. 26: GS and p-mTOR-S2448 are absent after Wnt-C59 treatment and DDC.IHC shows presence of both GS and p-mTOR around the central vein (cv) inWT at baseline. After DDC, expression of both proteins is decreased; thecombination of DDC and Wnt-C59 inhibition, however, leads to completeloss of GS and p-mTOR in zone 3.

DETAILED DESCRIPTION

The following description is merely exemplary in nature and is in no wayintended to limit the invention, its application, or uses. While thedescription is designed to permit one of ordinary skill in the art tomake and use the invention, and specific examples are provided to thatend, they should in no way be considered limiting. It will be apparentto one of ordinary skill in the art that various modifications to thefollowing will fall within the scope of the appended claims. The presentinvention should not be considered limited to the presently disclosedaspects, whether provided in the examples or elsewhere herein.

The use of numerical values in the various ranges specified in thisapplication, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges are both preceded by the word “about”. In this manner,slight variations above and below the stated ranges can be used toachieve substantially the same results as values within the ranges.Also, unless indicated otherwise, the disclosure of these ranges isintended as a continuous range including every value between the minimumand maximum values. For definitions provided herein, those definitionsrefer to word forms, cognates and grammatical variants of those words orphrases. As used herein “a” and “an” refer to one or more. Patentpublications cited below are hereby incorporated herein by reference intheir entirety to the extent of their technical disclosure andconsistency with the present specification.

As used herein, the terms “comprising,” “comprise” or “comprised,” andvariations thereof, are open ended and do not exclude the presence ofother elements not identified. In contrast, the term “consisting of” andvariations thereof is intended to be closed, and excludes additionalelements in anything but trace amounts.

As used herein, the term “patient” or “subject” refers to members of theanimal kingdom including but not limited to human beings and “mammal”refers to all mammals, including, but not limited to human beings.

As used herein, the “treatment” or “treating” of porphyria meansadministration to a patient by any suitable dosage regimen, procedureand/or administration route of a composition, device, or structure withthe object of achieving a desirable clinical/medical end-point,including but not limited to, preventing, reducing, and/or eliminatingany symptom of porphyria, such as liver damage, and/or reducing ordecreasing porphyrin production and/or accumulation. An amount of anyreagent or therapeutic agent, administered by any suitable route,effective to treat a patient is an amount capable of preventing,reducing, and/or eliminating any symptom of porphyria, such as liverdamage, and/or reducing or decreasing porphyrin production and/oraccumulation in a patient, e.g., ranging from 1 pg per dose to 10 g perdose, including any amount there between, such as 1 ng, 1 μg, 1 mg, 10mg, 100 mg, or 1 g per dose. The therapeutic agent may be administeredby any effective route, and, for example, may be administered as asingle bolus, at regular or irregular intervals, in amounts andintervals as dictated by any clinical parameter of a patient, orcontinuously.

The compositions described herein can be administered by any effectiveroute, such as parenteral, e.g., intravenous, intramuscular,subcutaneous, intradermal, etc., formulations of which are describedbelow and in the below-referenced publications, as well as isbroadly-known to those of ordinary skill in the art.

Active ingredients, such as nucleic acids or analogs thereof, may becompounded or otherwise manufactured into a suitable composition foruse, such as a pharmaceutical dosage form or drug product in which thecompound is an active ingredient. Compositions may comprise apharmaceutically acceptable carrier, or excipient. An excipient is aninactive substance used as a carrier for the active ingredients of amedication. Although “inactive,” excipients may facilitate and aid inincreasing the delivery or bioavailability of an active ingredient in adrug product. Non-limiting examples of useful excipients include:antiadherents, binders, rheology modifiers, coatings, disintegrants,emulsifiers, oils, buffers, salts, acids, bases, fillers, diluents,solvents, flavors, colorants, glidants, lubricants, preservatives,antioxidants, sorbents, vitamins, sweeteners, etc., as are available inthe pharmaceutical/compounding arts.

Useful dosage forms include: intravenous, intramuscular, orintraperitoneal solutions, oral tablets or liquids, topical ointments orcreams and transdermal devices (e.g., patches). In one embodiment, thecompound is a sterile solution comprising the active ingredient (drug,or compound), and a solvent, such as water, saline, lactated Ringer'ssolution, or phosphate-buffered saline (PBS). Additional excipients,such as polyethylene glycol, emulsifiers, salts and buffers may beincluded in the solution.

Suitable dosage forms may include single-dose, or multiple-dose vials orother containers, such as medical syringes, containing a compositioncomprising an active ingredient useful for treatment of porphyria asdescribed herein.

Expression of a gene refers to the conversion of a DNA sequence of agene, e.g., the CTNNB1 gene, to an active, mature gene product such as apolypeptide/protein, or a functional nucleic acid, and includes, forexample, transcription, post-transcriptional modification (e.g.,splicing) translation, and post-translational processing and/ormodification of a protein. Expression of a gene can be reduced by anyeffective mechanism at any stage of the gene expression process, such asby affecting transcriptional activation, transcription,post-transcriptional RNA processing, translation, and post-translationalprocessing or modification. Activity of a gene product, such asβ-catenin, may be decreased not only by decreasing expression of theactive protein product, but by affecting the mature protein product,such as by blocking, decoying, or otherwise interfering with the bindingof the active product, or a complex containing the active product, toprevent its activity, e.g., of β-catenin in Wnt/β-catenin/TCFtranscriptional activation.

Provided herein is a method of treating porphyria in a patient thatcomprises decreasing expression of β-catenin, or activity of β-cateninin a patient, e.g., in a patient's liver. There are a number of ways todecrease expression or activity of β-catenin in a patient, including,for example, and without limitation: RNA interference, antisensetechnology, and inhibition of the transcriptional activation activity ofβ-catenin through use of, e.g., small molecules or reagents thatinterfere with, e.g., Wnt/β-catenin/TCF transcriptional activationactivity, such as decoys, binding reagents, antagonists, etc., asdescribed in further detail below. Treatment of a patient results in adecrease in one or more symptoms of porphyria, such as liver damage, andresults in decrease of porphyrin production and/or accumulation in thepatient.

β-catenin (also Catenin beta-1, or CTNNB1, encoded in humans by theCTNNB1 gene, see, e.g., Gene ID: 1499, HGNC ID: HGNC:2514, OMIM 116806,UniProtKB—P35222 (CTNB1_HUMAN)) is an adherens junction protein and adownstream component of the canonical Wnt signaling pathway. Anexemplary, but non-limiting sequence for human β-catenin (SEQ ID NO: 1)is provided in FIG. 1.

Expression of β-catenin may be accomplished by RNA interference (RNAi),using an RNAi reagent, such as a nucleic acid or nucleic acid analog.United States Patent Application Publication No. 2016/0186176 A1describes various RNAi reagents, e.g., Dicer substrate siRNA (DsiRNA)reagents, including first and second nucleic acid strands and duplexes.The reagents are nucleic acids or modified nucleic acids or nucleic acidanalogs. Exemplary DsiRNAs, in reference to their designation in UnitedStates Patent Application Publication No. 2016/0186176 A1, include:βc-284, βc-288, βc-639, βc-830, βc-893, βc-894, βc-895, βc-900, βc-1306,βc-1310, βc-1314, βc-1541, βc-1545, βc-1566, βc-1567, βc-1568, βc-1569,βc-1652, βc-1662, βc-1667, βc-1681, βc-1682, βc-1683, βc-1820, βc-2097,βc-2144, βc-2151, βc-2277, βc-2350, βc-2442, βc-2445, βc-2517, βc-2521,βc-2525, βc-2611, βc-2612, βc-2620, βc-3111, βc-3195, βc-3389, βc-3393,βc-3399, βc-3500, βc-3534, βc-3589, βc-3591, βc-3653, βc-3659, βc-3708,βc-3712, βc-240, βc-244, βc-253, βc-259, βc-264, βc-496, βc-516, βc-540,βc-582, βc-686, βc-692, βc-697, βc-707, βc-753, βc-870, βc-889, βc-1060,βc-1070, βc-1154, βc-1180, βc-1412, βc-1418, βc-1579, βc-1620, βc-1816,βc-2282, βc-3203, βc-3333, βc-3354, βc-3426, βc-3431, βc-3605, βc-3615,βc-3674, βc-3686, βc-3691, βc-m1354, βc-m1515, βc-m1763, βc-m2568,βc-m2806, βc-m3092, βc-m3207, βc-m3444, βc-m3449, or βc-m3533, in oneaspect they include βc-1545, βc-1683, βc-2097, βc-2277, βc-2612,βc-3111, βc-3195, βc-3389, βc-3393, βc-3399, βc-3534, βc-3653, βc-3659,βc-3708, βc-3712, βc-253, βc-259, βc-686, βc-692, βc-697, βc-870,βc-889, βc-1154, βc-1180, βc-1412, βc-2282, βc-3203, βc-3431, βc-m1763,βc-m2806, βc-m3207, or βc-m3533, and in one aspect, they are e.g.,βc-1545, βc-1683, βc-3195, βc-3389, βc-3393, βc-3399, βc-3534, βc-3659,βc-3712, βc-253, or βc-3203, and in a further aspect, they includeDsiRNAs of Table 18 of US 2016/0186176 A1, as follows:

(SEQ ID NO: 2) 5′-CAGGGATTTTCTCAGTCCTTCACTCAA-3′; (SEQ ID NO: 3)5′-TGATGGACAGTATGCAATGACTCGAGC-3′; (SEQ ID NO: 4)5′-TGCTGCTCATCCCACTAATGTCCAGCG-3′; (SEQ ID NO: 5)5′-GCAGAATACAAATGATGTAGAAACAGC-3′; (SEQ ID NO: 6)5′-CACCTGTGCAGCTGGAATTCTTTCTAA-3′; (SEQ ID NO: 7)5′-TGAACTTGCTCAGGACAAGGAAGCTGC-3′; (SEQ ID NO: 8)5′-TAGCCTTGCTTGTTAAATTTTTTTTTT-3′; (SEQ ID NO: 9)5′-GTAGAACACTAATTCATAATCACTCTA-3′; (SEQ ID NO: 10)5′-TAAATCAGTAAGAGGTGTTATTTGGAA-3′; (SEQ ID NO: 11)5′-AAAAATGGTTCAGAATTAAACTTTTAA-3′; (SEQ ID NO: 12)5′-CAGGGATTTTCTCAGTCCTTC-3′; (SEQ ID NO: 13)5′-TGATGGACAGTATGCAATGAC-3′; (SEQ ID NO: 14)5′-TGCTGCTCATCCCACTAATGT-3′; (SEQ ID NO: 15)5′-GCAGAATACAAATGATGTAGA-3′; (SEQ ID NO: 16)5′-CACCTGTGCAGCTGGAATTCT-3′; (SEQ ID NO: 17)5′-TGAACTTGCTCAGGACAAGGA-3′; (SEQ ID NO: 18)5′-TAGCCTTGCTTGTTAAATTTT-3′; (SEQ ID NO: 19)5′-GTAGAACACTAATTCATAATC-3′; (SEQ ID NO: 20)5′-TAAATCAGTAAGAGGTGTTAT-3′; and (SEQ ID NO: 21)5′-AAAAATGGTTCAGAATTAAAC-3′,or modified nucleic acids or nucleic acid analogs of any of thepreceding, such as 2′-O-methyl-modified versions thereof.

In aspects, antisense reagents also may be used to knock down β-cateninexpression (see, e.g., U.S. Pat. No. 6,066,500 and Popov, V. B., et al.,Second-generation antisense oligonucleotides against β-catenin protectmice against diet-induced hepatic steatosis and hepatic and peripheralinsulin resistance, FASEB J. 2016; 30(3)1207-1217).

In aspects, inhibitors, such as small molecule inhibitors of theactivity of β-catenin also may be used to decrease β-catenin activityand thereby treat porphyria. In aspects, such inhibitors inhibitWnt/β-catenin/TCF transcriptional activation activity. In one aspect theinhibitor is ICG001 or an isomer, stereoisomer, or enantiomer thereof,including pure or racemic mixtures of stereoisomers of ICG001.

ICG001 and related compounds are described in U.S. Pat. No. 8,293,743.PRI 724 is an enantiomer of ICG001, as seen in FIG. 2.

In further aspects, inhibitors of the activity of β-catenin includeporcupine (PORCN) inhibitors, which also may be used to decreaseβ-catenin activity and thereby treat porphyria. PORCN is required forthe synthesis of Wnt ligands, which participate in the Wnt signalingpathway. PORCN is a membrane-bound O-acyltransferase located in theendoplasmic reticulum of cells and mediates the palmitolyation/acylationof serine/threonine residues of the Wnt ligand. Successfulpalmitolyation/acylation causes the secretion of Wnt ligands andactivates Wnt-mediated signaling through the accumulation of β-cateninin the cytoplasm of the cell. β-catenin is eventually translocated tothe nucleus, where its accumulation drives the downstream regulation ofgene expression. The dysregulation of this Wnt signaling pathway canlead to tumor cell proliferation and injury. The administration of PORCNinhibitors prevents the palmitolyation/acylation of the Wnt ligand,which prevents β-catenin accumulation. Without the Wnt ligand, β-cateninis phosphorylated in the cell cytoplasm and is degraded. Thus, lack ofaccumulation of β-catenin in the nucleus of the cell disrupts the Wntsignaling pathway.

A number of PORCN inhibitors (FIGS. 3A-3C) are known, including, withoutlimitation: LGK974 (see, e.g., U.S. Pat. No. 8,546,396 B2 and Liu J, etal., Targeting Wnt-driven cancer through the inhibition of Porcupine byLGK974. Proc. Natl. Acad. Sci. U.S.A 2013; 110(50):20224-20229); CGX1321(see, e.g., International Patent Application Publication No. WO2014/165232 A1, United States Patent Application Publication No.2018/0153884 A1, U.S. Pat. No. 10,238,652 B2, and Jiang, J., et al. Anovel porcupine inhibitor blocks WNT pathways and attenuates cardiachypertrophy. Biochim. Biophys. Acta, Mol. Basis Dis. 2018;1864(10):3459-3467); Wnt-C 59 (see, e.g., U.S. Pat. No. 8,546,396 B2 andProffitt, K. D., et al. Pharmacological Inhibition of the WntAcyltransferase PORCN Prevents Growth of WNT-Driven Mammary Cancer.Cancer Res. 2013; 73(2):502-507); ETC-1922159 (ETC-159) (see, e.g., U.S.Pat. No. 9,926,320 B2 and Madan, B., et al. Wnt addiction of geneticallydefined cancers reversed by PORCN inhibition. Oncogene 2016;35:2197-2207); IWP2 and IWP1 (see, e.g., U.S. Pat. No. 9,783,550 B2 andChen, B., et al. Small molecule-mediated disruption of Wnt-dependentsignaling in tissue regeneration and cancer. Nat. Chem. Biol. 2009;5(2):100-107); IWP-O1 (see, e.g., You, L., et al. Development of atriazole class of highly potent Porcn inhibitors. Bioorg. Med. Chem.Lett. 2016; 26(24):5891-5895); RXC004 (see, e.g., U.S. Pat. No.10,202,375 B2, U.S. Pat. No. 10,047,079 B2, and Bhamra, I., et al. NovelPorcupine (PORCN) inhibitor RXC004: Evaluation in models of RNF43 lossof function cancers. J. Clin. Oncol. 2017; 35(15)); IPW-3 and IPW-4(see, e.g., U.S. Pat. No. 9,783,550 B2); GNF-6231 (see, e.g., U.S. Pat.No. 8,546,396 B2, and Cheng, D., et al. Discovery of Pyridinyl AcetamideDerivatives as Potent, Selective, and Orally Bioavailable PorcupineInhibitors. ACS Med. Chem. Lett. 2016; 7:676-680); IWP-12 (see, e.g.,U.S. Pat. No. 9,783,550 B2 and Wang, X., et al. The Development ofHighly Potent Inhibitors of for Porcupine. J. Med. Chem. 2013;56(6):2700-2704); and IWP-L6 (see, e.g., U.S. Pat. No. 9,783,550 B2 andWang, X. et al. J. Med. Chem. 2013; 56(6):2700-2704). InternationalPatent Application Publication No. WO 2014/165232 A1 describes a numberof PORCN inhibitors.

Reagents, such as siRNA, DsiRNA, antisense reagents, and small moleculessuch as PRI-724 or ICG001, can be targeted to a target organ, such asthe liver, by conjugation to a targeting moiety, such as N-acetylgalactosamine (GaINAc) by a hydrolyzable bond such as an ester, which iscleaved in vivo, releasing the reagent locally. GaINAc, e.g.,triantennary GaINAc, has been used to target oligonucleotides to theliver (Prakash, T. P., et al. Targeted delivery of antisenseoligonucleotides to hepatocytes using triantennary N-acetylgalactosamine improves potency 10-fold in mice, Nucl. Acids Res. 2014;42(13):8796-8807).

We have shown that mice lacking β-catenin in hepatocytes andcholangiocytes (albumin-cre β-catenin knockout (KO) mice; KO1) had fewerA6-positive atypical ductular cells in response to DDC (Apte, U., et al.Wnt/beta-catenin signaling mediates oval cell response in rodents.Hepatology 2008; 47:288-295), which was not due to decreased levels oftotal liver bile acids (Thompson, M. D., et al. Beta-catenin regulationof farnesoid X receptor signaling and bile acid metabolism during murinecholestasis. Hepatology 2018; 67(3):955-971). In the current study, wedemonstrate that mice lacking Wnt/β-catenin pathway components orfollowing pharmacologic β-catenin inhibition exhibited less hepaticinjury after DDC. This was due to a novel role of Wnt/β-catenin pathwayin regulating heme biosynthesis, which resulted in lesser porphyrinaccumulation, fewer protein aggregates, and lesser overall hepaticinjury. Thus, inhibiting Wnt/β-catenin signaling may represent apotential therapeutic strategy for patients with hepatic porphyria.

Example 1 Materials and Methods

Animals.

Male mice with β-catenin loxp+/+ or LRP5/6 loxp+/+ were backcrossed toAlb-Cre+/− (all in a C57BL6 background) to generate β-catenin KO mice(KO1) or LRP5/6 double KO mice (KO2) in hepatocytes and cholangiocytes.Littermates with floxed alleles but without Cre were used as respectivewildtype controls (WT1, WT2). Mice were fed a diet containing 0.1% DDC(Bioserve, Frenchtown, N.J.) at 2 months of age for 14 days, asdescribed previously. On the 15th day animals were sacrificed. Bloodsamples were collected from the inferior vena cava and serum isolatedfor biochemical analysis. Portions of the liver were fixed in 10%formalin and processed for paraffin embedding; the remaining liver wasfrozen in liquid nitrogen and stored at −80° C.

Therapeutic Intervention with β-Catenin DsiRNA LNP.

Two-month old male CD-1 mice were started on 0.1% DDC. Three days afterstarting diet, mice received either one intravenous (i.v.) injection ofβ-catenin Dicer-substrate siRNA (DsiRNA) formulated in a lipidnanoparticle (LNP) from Dicerna Pharmaceuticals (Watertown, Mass.) at 3mg/kg, or scrambled (control) DsiRNA LNP i.v. at the same concentration.Mice were dosed weekly thereafter with either control or β-cateninDsiRNA until the time of sacrifice (day 31 after the start of DDC).Livers from the control and experimental groups were utilized forimmunohistochemistry (IHC), Western blotting, and biochemical assays.

Serum Biochemistry.

Serum biochemical measurements were performed by the University ofPittsburgh Department of Pathology Laboratory Support Services. Totalbilirubin, direct bilirubin, alkaline phosphatase (ALP), aspartateaminotransferase (AST), and alanine aminotransferase (ALT) were measuredin samples taken before sacrifice at multiple time points.

Immunohistochemistry.

Tissues fixed in 10% formalin were embedded in paraffin, and 4-μmsections cut onto Superfrost Plus glass slides (Thermo FisherScientific, Pittsburgh, Pa.) were used for H&E, Sirius Red, orimmunohistochemical analysis as described elsewhere1. Primary antibodiesused include β-catenin (1:100, sc-7199), glutamine synthetase (GS; 1:50,sc-9067), cyclin D1 (1:50, sc-753), and CD45 (1:100, sc-53665) fromSanta Cruz Biotechnology, Dallas, Tex.; Ki67 (1:100, RM9106-S) fromThermo Fisher Scientific; CD68 (1:100, MCA1957) and F4/80 (1:100,MCA497GA) from Bio-Rad, Hercules, Calif.; Neutrophil Elastase (1:1500,ab68672) and Sox9 (1:100, ab5535) from Abcam, Cambridge, Mass.; andEpCAM (1:10, Clone G8.8) from Developmental Studies Hybridoma Bank,University of Iowa. Apoptosis was determined using terminaldeoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining(ApopTag Peroxidase in Situ Apoptosis Detection Kit, Millipore,Temecula, Calif., USA).

Quantification of Staining

Polarized light images were taken with an Olympus Provis microscope.Each image was split into red, green and blue channels using ImageJFiji, among which the red channel was chosen since it has the bestseparation. Next the staining was isolated by using Threshold setting 25for the upper level and 255 for the lower level. Then the percentage ofthe stained area to the total image was measured.

Bright field images of H&E staining were split into hematoxylin, eosinand DAB channels using ImageJ Fiji. The color of porphyrin plugs wasshown in the DAB channel. Next, the color of porphyrin plugs wasisolated by using Threshold setting 0 for the upper level and 75 for thelower level. Then the percentage of the porphyrin area to the totalimage was measured.

Protein Extraction and Western Blot Analysis.

Approximately 20 mg of liver was dounce homogenized in a buffercontaining 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% Nonidet P-40, 1%sodium deoxycholate, 0.1% SDS (RIPA buffer) supplemented with proteaseinhibitor as described previously (Ajioka, R. S., et al. Biosynthesis ofheme in mammals. Biochim. Biophys. Acta 2006; 1763:723-736). Proteinassays were performed using bicinchoninic acid protein assay. Equalamounts of protein (10-50 μg) were resolved on Tris-HCl precast gels(Bio-Rad) by SDS-PAGE analysis using the Mini-PROTEAN 3 ElectrophoresisModule Assembly (Bio-Rad). The resolved proteins were transferred topolyvinylidene difluoride membranes, followed by immunoblotting andvisualization by enhanced chemiluminescence. The primary antibodies usedwere mouse antibody to β-catenin (1:1000, BD610154, BD Biosciences, SanJose, Calif.); total YAP (1:1000, 4912), Phospho-YAP Ser127 (1:1000,4911), CHOP (1:2000, 2895), and IRE1α(1:2000, 3294) from Cell SignalingTechnology, Los Angeles, Calif.; cyclin D1 (1:200, RB-9041-PO) fromThermo Fisher Scientific; GAPDH (1:400, sc-25778), GS (1:1000,sc-74430), ferrochelatase (1:100, sc-377377), ubiquitin (Ub; 1:2000,sc-8017), and lamin A/C (1:2000, sc-37624) from Santa Cruz;hydroxymethylbilane synthase (HMBS; 1:500, MBS9407926) from MyBioSource,San Diego, Calif.; Cyp3a1 (1:2000, ab22724), GST mu (1:2000, ab53942),and lamin B1 (1:2000, ab16048) from Abcam; aminolevulinic acid synthase(ALA-S; 1:1000, NB100-56415) from Novus Biologicals, Littleton, Colo.);aminolevulinic acid dehydratase (ALA-D; 1:1000, AV41657) from Sigma; andkeratin 8 (1:2000, Troma) and keratin 18 (1:2000, Ab-46683) fromDevelopmental Studies Hybridoma Bank. Horseradish peroxidase conjugatedsecondary antibodies were purchased from Millipore (Billerica, Mass.).

Porphyrin Measurement from Total Liver Homogenate.

Porphyrin was measured in the liver lysate in the form of totalfluorescent porphyrin content (including Uro, Copro, PP-IX, and NMP)utilizing the intrinsic fluorescence of the demetallated porphyrins.Briefly, 2 μl of the lysate was added to 200 μl of 1:1 mixture ofethanol:perchloric acid (0.9 N). The fluorescence of the resultingsolution was measured in a Biotek Synergy HT 96-well plate reader usingthe filter sets 400/30 nm (excitation) and 590/35 nm (emission). Theamount of total porphyrins was expressed as fluorescent intensitynormalized to the protein content of the sample.

In-Gel Heme Staining.

Protein bound heme was detected utilizing a modification of a previouslypublished method (Smith, A. G., et al. Drugs and the hepatic porphyrias.Clin. Haematol. 1980; 9:399-425). Briefly, frozen livers werehomogenized in phosphate buffered saline (PBS) containing 1% Empigen BB,5 mM EDTA, and supplemented with protease inhibitor cocktail. 100 μg oftotal protein was separated by reducing SDS-PAGE, or non-denaturingPAGE, in 4° C. at 60V, and the proteins were transferred to a PVDFmembrane. After transfer the membrane was washed with PBS twice, andthen visualized with enhanced chemiluminescence reagent andautoradiograph film.

Metabolomic Analysis.

Liver samples were homogenized in water (100 mg tissue in 500 uL water),and then a 200 uL aliquot of methanol: acetonitrile (v/v, 1:1) was addedto 100 uL of liver homogenate. The mixture was vortexed twice for 1 minand centrifuged at 15,000 g for 20 minutes. One microliter of thesupernatants from all samples was injected onto the ultra-performanceliquid chromatography and quadrupole time-of-flight mass spectrometry(UPLC-QTOFMS) for analysis as described previously (Tephly, T. R., etal. Studies on the mechanism of experimental porphyria andferrochelatase inhibition produced by3,5-diethoxycarbonyl-1,4-dihydrocollidine. Int. J. Biochem. 1980;12:993-998).

Ferrochelatase Activity Assay.

Activity of ferrochelatase in control DsiRNA and β-cateninDsiRNA-treated livers after DDC was performed as previously described6.Briefly, mitochondria were isolated from mouse livers treated with DDCand either control or β-catenin DsiRNA. Mitochondrial membranes wereisolated and suspended in 1.0% sodium cholate and 0.1 M KCl. The mixturewas centrifuged to separate the solubilized enzyme from membranefragments, and saturated ammonium sulfate added to the enzyme. Aftercentrifugation and further fractionation by saturated ammonium sulfate,the resulting pellet was dissolved in 1% Triton X-100 and 0.5 M KCl andthen applied to a sepharose column. Ferrochelatase was eluted from thecolumn with 1% sodium cholate and 1.5 M KCl. Fractions exhibitingferrochelatase activity were incubated with 0.1 mM porphyrin in 50 mMTris acetate, pH 8.1, 5 mM dithiothreitol, 0.2 mM ferrous ammoniumcitrate, and 0.2% Triton X-100 at 37° C. in the dark for 30 minutes. Thereaction was terminated by addition of 50 mM iodoacetamide, and theproduct quantitated with a spectrophotometer.

Measurement of Liver Bile Acids.

Liver total bile acids were measured using a total bile acids kit fromCrystal Chem (Downers Grove, Ill.), as per the manufacturer'sinstructions. Briefly, frozen liver tissue was homogenized in 70%ethanol at room temperature and then incubated in tightly capped glasstubes at 50° C. for 2 hours. The homogenates were centrifuged at 6,000 gfor 10 minutes to remove debris. Total bile acid levels were measuredand concentrations determined using the calibration curve and meanchange in absorbance value for each sample.

RNA Isolation and Real-Time PCR.

RNA was extracted from frozen livers using Trizol (Invitrogen). RNA wasDNase-treated and equal microgram amounts of RNA from each sample wereused to make individual cDNA samples with SuperScript III First StrandSynthesis System for RT-PCR (Invitrogen). cDNA along with 1× PowerSYBR-Green PCR Master Mix (Applied Biosystems) and the appropriateprimers were used for each real-time PCR reaction. The AppliedBiosystems StepOnePlus Real-Time PCR System was used for the analysis ofthe transcripts with the StepOne v2.1 software. The comparative ΔΔCTmethod was used for analysis of the data and all data is presentednormalized to WT at baseline. GAPDH expression was used as the internalcontrol.

cDNA Arrays.

Livers from control and β-catenin DsiRNA LNP treatment were harvested 31days after the start of DDC and mRNA was isolated using TRIzol(Invitrogen, Carlsbad, Calif.). mRNA was then analyzed forNF-κB-regulated gene expression with a mouse NF-κB-regulated cDNA platearray (Signosis, Sunnyvale, Calif.) as per the manufacturer'sinstructions. Briefly, mRNA was reverse transcribed using abiotin-labeled NF-κB primer mix, mixed with hybridization buffer, andadded to a plate containing 21 target genes. The plate was thenincubated at 450 C overnight, washed the next day, blocked, anddeveloped with substrate. The plate was read on a BioTek HT (BioTek,Winooski, Vt.) with no filter to detect luminescence. The data werenormalized to control (WT) values and then plotted on a bar graph.

Transcription Factor Assay.

Nuclear lysates from control and β-catenin DsiRNA LNP treated liversharvested 31 days after starting DDC were analyzed for NF-κB activityusing the NF-κB p65 EZ-TFA Transcription Factor Assay (Millipore,Billerica, Mass.) as per the manufacturer's instructions. Briefly, 15 μgof each sample was added to the plate and incubated with blockingreagent for 1.5 hours at room temperature. The plate was washed,incubated with NF-κB antibody provided by the manufacturer, washed andincubated with rabbit secondary antibody, and then developed withsubstrate. The plate was read on a BioTek HT at an absorbance of 450 nm.

Characterization of the ALA-D Promoter Region.

ChIPseq studies of RXRα, FXR, TCF4, HNF4α, LXR, PPAR, CAR, H4K5Ac(transcriptionally active chromatin), and H3K4Me3 (active promoterregions) were aligned with RNAseq reads to identify binding regions.Data is derived from a manuscript in preparation by Tian and Locker; theFXR analysis is described in Correia, M. A., et al. Cytochrome P450regulation: the interplay between its heme and apoprotein moieties insynthesis, assembly, repair, and disposal. Drug Metab. Rev. 2011;43:1-26.

Statistical Analysis.

All experiments were performed with three or more animals, andrepresentative data are presented. Quantification of positive cells andserum biochemistry measurements were compared for statistical analysisby Student's t-test (Graphpad 6.0), and P<0.05 or 0.01 was consideredsignificant or extremely significant, respectively.

Results

β-Catenin KO Mice have Less Injury and Inflammation than WT after 14Days of DDC.

KO1 and WT1 littermates were fed DDC diet for 14 days and assessed forextent of injury (FIG. 4A). Gross examination revealed black-brown andenlarged WT1 livers, which is characteristic of DDC, while KO1 liverswere smaller and normal looking (FIG. 4B). Likewise, KO1 mice showeddecreased liver weight/body weight ratio. Notably, both hepatocellularand biliary injury are decreased in KO1, as shown by significantly lowerserum alkaline phosphatase (ALP) and alanine aminotransferase (ALT)(FIG. 4C).

Immunohistochemistry (IHC) for glutamine synthetase (GS), atranscriptional target of β-catenin, as well as WB for β-catenin,confirms β-catenin loss from hepatocytes in KO1 (FIG. 4D; FIG. 5A).Histology reveals KO1 livers have a noticeable decrease in dark brownpigmentation indicative of porphyrin accumulation compared to WT1 (FIG.4E). However, fibrosis, as assessed by Sirius red, was equivalent inboth genotypes (FIG. 4F).

By IHC, we found significantly fewer cyclin D1 positive cells in KO1livers due to loss of β-catenin, which was confirmed by WB (FIG. 5A;FIG. 6A). KI67 IHC also showed less hepatocyte proliferation in KO1after DDC; however, cholangiocyte proliferation was still evidentdespite β-catenin loss (FIG. 6A). We next stained WT1 and KO1 liverswith EpCAM and Sox9 and found equivalent ductular response in bothphenotypes (FIG. 6B). Additionally, although total Yap protein wasincreased in KO1, the phosphorylated, inactive Yap increased as well,suggesting ductular mass is comparable in WT1 and KO1 (FIG. 5A).

Finally, we examined hepatic inflammation in both groups after DDC. Weobserved a decrease in both CD68- and CD45-positive inflammatory cellsin KO1 when compared to WT1 (FIG. 6C). There were also fewerelastase-positive neutrophils infiltrating the parenchymal tissue inperiportal region. F4/80-stained macrophages were comparable in bothgroups. These findings suggest that on the whole, inflammation is lessin KO1 compared to WT1 mice.

Mice Lacking Wnt Signaling Show a Similar Protective Phenotype asβ-Catenin KO after DDC Feeding.

Next, we assessed whether loss of upstream Wnt signaling also protectsmice from DDC-induced liver injury. LRP5/6, a co-receptor for Wnt, isnecessary to activate the canonical Wnt/β-catenin signaling pathway.LRP5/6-double KO mice (KO2) and littermate controls (WT2) were fed DDCfor 14 days (FIG. 7A). KO2 mice had noticeably lighter-colored, smallerlivers and decreased liver weight/body weight ratios compared with WT2mice (FIG. 7B; FIG. 7C). In serum, ALP was significantly lower in KO2than WT2 after DDC, with ALT approaching significance (FIG. 7D),indicating that like KO1, KO2 mice have less injury.

KO2 mice lack active β-catenin as confirmed by IHC for GS, despite thepresence of hepatic β-catenin (FIG. 7E; FIG. 5B). H&E staining showsthat like KO1, KO2 have decreased porphyrin accumulation compared toWT2, with comparable fibrosis (FIG. 7F; FIG. 7G). Interestingly,although cyclin D1 expression is dramatically decreased in KO2 afterDDC, as demonstrated by both IHC and WB (FIG. 8A; FIG. 5B),Ki67-positive hepatocytes are still present in KO2 (FIG. 8A), suggestingWnt/p-catenin-independent activation of proliferation. Similar to KO1,cholangiocyte proliferation is equivalent in WT2 and KO2, concomitantwith equivalent Yap expression, indicating that β-catenin activation isnot required for biliary repair after DDC (FIG. 5B; FIG. 8B). We alsoobserved inflammatory markers CD45, F4/80, and elastase were decreasedin KO2, while CD68 was equivalent in both genotypes (FIG. 8C). Insummary, KO2 mice phenocopy key features of KO1 on DDC, includingdecreased biliary injury, inflammation, and porphyrin accumulation.

Exogenous inhibition of β-catenin using DsiRNA formulated in a lipidnanoparticle recapitulates the protective phenotype seen in β-cateninand LRP5/6 KO on DDC.

To address the relevance of therapeutic intervention, we next suppressedβ-catenin exogenously in DDC-induced injury. We administered 3 mg/kgcontrol or β-catenin DsiRNA formulated into a lipid nanoparticle (LNP)to mice once weekly beginning 3 days after starting DDC; the CD-1 strainwas used in these studies to assure rigor and reproducibility (FIG. 9A).WB shows successful knockdown of β-catenin protein (FIG. 9B), and thatthe LNP preferentially targets the DsiRNA to hepatocytes (FIG. 10A). Asin KO1 and KO2, livers of mice treated with β-catenin DsiRNA werenotably smaller and lighter in color, with significantly decreased liverweight/body weight ratio (FIG. 9C). Both ALP and ALT were alsosignificantly decreased after β-catenin DsiRNA administration (FIG. 9D),although overall injury was lesser compared to WT1 and comparable toWT2, likely due to strain-specific differences. The reduced injury afterβ-catenin DsiRNA treatment also resulted in a notable and significantdecrease in fibrosis, as assessed by Sirius red staining and ImageJquantification (FIG. 9E), although a-smooth muscle actin-positivemyofibroblasts were evident in both control and β-catenin DsiRNA-treatedlivers (FIG. 10B). Mice treated with β-catenin DsiRNA also had decreasedhepatocyte proliferation (FIG. 10C). CD45- and F4/80-positive cells weredecreased after β-catenin DsiRNA, while elastase IHC shows equivalentneutrophil accumulation in both treatment groups (FIG. 10D), indicatingthat as in KO1 and KO2, exogenous β-catenin suppression alters theinflammatory response after DDC.

Interestingly, the ductular response in the β-catenin DsiRNA-treatedlivers extended from the periportal region into the parenchyma, as shownby Sox9 staining (FIG. 10E). Despite the expansion of these atypicalductules, we did not detect an increase in either Notch or Yap signalingin the absence of β-catenin (FIG. 11A; FIG. 11B), indicating that lossof β-catenin does not alter the ductular response.

Protection from Injury is Independent of NF-κB Activation or Bile AcidAccumulation.

We previously reported that β-catenin KO mice are protected fromTNF-α-induced apoptosis and bile acid induced injury through activationof NF-κB and farnesoid X receptor (FXR), respectively (Thompson, M. D.,et al. Beta-catenin regulation of farnesoid X receptor signaling andbile acid metabolism during murine cholestasis. Hepatology 2018;67(3):955-971 and Nejak-Bowen, K., et al. Beta-catenin-NF-kappaBinteractions in murine hepatocytes: a complex to die for. Hepatology2013:57:763-774); thus, we wanted to determine whether either of thesemechanisms were playing a role in protection from DDC-induced injury inthe absence of β-catenin. Transcription factor assay showed a mildincrease in NF-κB DNA binding activity after DDC, which was not furtherincreased with β-catenin DsiRNA (FIG. 12A). We have also previouslyshown that 4 weeks of DDC induces only a modest increase in bile acidsin WT1 mice which was insignificantly decreased in KO1 (Thompson, M. D.,et al. Beta-catenin regulation of farnesoid X receptor signaling andbile acid metabolism during murine cholestasis. Hepatology 2018;67(3):955-971). However, the amount of total bile acids in the liver wasnot significantly different between control DsiRNA and β-catenin DsiRNAtreatment groups after DDC (FIG. 12B). Therefore, neither increasedcytoprotection nor decreased bile acid-induced injury explains theprotection from injury seen after DDC in the β-catenin DsiRNA-treatedgroup.

β-Catenin DsiRNA Treatment Reduces the Amount of Protoporphyrin inLivers after DDC Compared to Control DDC.

One of the remarkable features of livers treated with β-catenin DsiRNAand DDC was a decrease in porphyrin accumulation, which gives theDDC-treated livers their characteristic dark-brown color. Similarfindings were also noted in livers of KO1 and KO2 mice on DDC (FIG. 4F;FIG. 13F). Whereas in control DsiRNA-treated livers porphyrin wasevident throughout the parenchyma as well as in the periportal region,in β-catenin DsiRNA treated livers, only a few porphyrin plugs wereevident, and these were exclusively located within the bile ductules(FIG. 13A). Quantification of porphyrin accumulation by channelsplitting of brightfield images confirms this decrease (FIG. 13B).Furthermore, when total liver homogenates were assayed for porphyrin andthe porphyrin fluorescence normalized to total protein, a 3-foldreduction in total porphyrin was evident in mice treated with β-cateninDsiRNA and DDC compared to mice treated with control DsiRNA and DDC(FIG. 13C). Using liquid chromatography-mass spectrometry (LC-MS) as anadditional modality of measuring porphyrin content, we observed thatPP-IX levels are significantly less in livers of β-catenin DsiRNAtreated mice after DDC (FIG. 13D). Thus, inhibition of β-cateninprevents porphyrin accumulation after DDC.

Alterations in Expression of Cytochrome P450 Enzymes and CAR in theAbsence of β-Catenin do not Contribute Significantly to Protection fromDDC.

To determine if detoxification plays a role in decreasing hepatic injuryafter DDC in mice lacking β-catenin, we examined mRNA levels of twoCyp3a isoforms, Cyp3a1 and Cyp3a11, at baseline and found a significantincrease in both in KO1 livers compared to WT1 (FIG. 14A). However,levels of these Cyp3a isoforms are unchanged in mice treated withβ-catenin DsiRNA at baseline before DDC (FIG. 14B), suggesting that theupregulation seen in KO1 may be a compensatory response. Althoughβ-catenin DsiRNA livers showed a modest increase in Cyp3a proteinexpression compared to control DsiRNA in the presence of DDC (FIG. 14C),it is unclear if this indicative of increased enzymatic activity orrather due to lack of Cyp degradation in these mice.

DDC also activates the constitutive androstane receptor (CAR), whichcontributes to hepatic injury. β-catenin regulates CAR expression, anddeletion of β-catenin in KO1 mice results in loss of CAR at both themRNA and protein level. Similarly, knockdown of β-catenin with DsiRNAsignificantly decreases CAR expression at baseline (FIG. 14D).Interestingly, CAR expression is suppressed in controls as a response toDDC, while DDC further decreases CAR in β-catenin DsiRNA treated mice.

To rule out if increased Cyp3a and decreased CAR expression after DDC inmice lacking hepatocyte β-catenin is interfering with DDC metabolism andhence is model-specific, we performed an activity assay onferrochelatase (FC) isolated from DDC-fed mice after control DsiRNA andβ-catenin DsiRNA treatment. Notably, there was no measurable activity ofFC in either group after DDC treatment (not shown), despite positivecontrols demonstrating the validity of the assay and confirming thenegative results. Thus, despite alterations in pathways that regulateDDC metabolism, these are not the primary mechanism by which these miceevade profound injury, as β-catenin-inhibited mice are responsive to DDCtoxicity as seen by complete inhibition of FC.

Decreased Porphyrin Accumulation after β-Catenin DsiRNA Treatment is aResult of Decreased Expression of Enzymes Involved in Heme Biosynthesis.

We next determined if upstream inhibition of the heme biosynthesispathway is responsible for the lack of protoporphyrin accumulation inβ-catenin-inhibited animals. We first examined the expression of ALA-S,the rate limiting step in heme biosynthesis, in controls with or withoutDDC and in β-catenin DsiRNA-treated DDC livers by WB. There was a markeddecrease in ALA-S protein in livers treated with β-catenin DsiRNA afterDDC, in contrast to livers with control DsiRNA, which had greater ALA-Sthan WT baseline samples FIG. 15A). Protein levels of FC, whichcatalyzes the final step in heme biosynthesis, were unchanged aftereither DDC or administration of either DsiRNA, despite its absentactivity (FIG. 15B). We also examined expression of several otherenzymes in the heme biosynthesis pathway, specifically δ-aminolevulinicacid dehydratase (ALA-D), which synthesizes porphobilinogen, and HMBS,which catalyzes the synthesis of hydroxymethylbilane. While levels ofHMBS were unchanged, ALA-D, like ALA-S, was decreased after β-cateninDsiRNA treatment (FIG. 15C).

To determine whether these enzymes were transcriptionally regulated byβ-catenin, we analyzed mRNA expression from livers treated with controlor β-catenin DsiRNA in the absence of DDC. Notably, both ALA-S and ALA-Dwere significantly suppressed in the presence of β-catenin DsiRNA (FIG.15D). Similar decreases in the mRNA expression of these enzymes werealso noted in both KO1 and KO2 at baseline (FIG. 15E; FIG. 15F).Furthermore, a strong TCF4 binding site was identified in the intronregion of the ALA-D gene by ChIP-seq (FIG. 16), which also correspondsto the binding sites for several other nuclear receptors, like FXR andHNF4. These data indicate that ALA-D may be a direct Wnt/β-catenintarget, and that suppression of this enzyme inhibits the early steps inthe heme biosynthesis pathway, potentially preventing the accumulationof protoporphyrins.

Loss of β-Catenin Blunts the Ability of DDC to Deplete Heme.

One of the major pathways by which DDC mediates its porphyrinogeniceffect is through heme depletion, which inhibits the function ofhemoproteins such as Cyps that can metabolize and detoxify DDC. Becauseβ-catenin DsiRNA treated livers had less porphyrin accumulation afterDDC feeding, we determined whether loss of protein bound heme after DDCfeeding is decreased in β-catenin inhibited mice compared to mice on DDCalone. We assayed for heme content in hepatic heme-containing proteinsand found DDC feeding caused massive heme depletion (FIG. 17A (panel A);FIG. 17A (panel B)). However, upon longer exposure of the film, there isresidual protein-bound heme apparent in β-catenin DsiRNA treated liversafter DDC feeding compared to controls on DDC (FIG. 17A (panel C); FIG.17A (panel D)), indicating lesser DDC-mediated heme destruction. Asimilar trend was observed in denaturing PAGE (FIG. 18 (panels A, B, C,and D). We also measured heme levels in the livers by LC-MS and foundpreservation of total heme (free and protein-bound) in β-catenininhibited livers after DDC (FIG. 17B). The increased heme content in thelivers of β-catenin DsiRNA treated mice relative to control DDC micecould also be a function of decreased heme degradation, which iscatalyzed by heme oxygenase (HO1). We found HO-1 expression wassignificantly repressed in β-catenin DsiRNA treated livers, both beforeand after DDC (FIG. 17C). Thus, heme depletion as a consequence of DDCis blunted in the absence of β-catenin through multiple mechanisms.

Loss of β-Catenin Decreased Protein Aggregation after DDC.

We next analyzed the extent of protein aggregation after DDC. SDS-PAGEand Coomassie blue staining shows changes in the protein banding patternas a function of DDC (FIG. 19). Firstly, there is a loss of discretebanding patterns after porphyrin accumulation (compare bands in positioniii, iv, v in DDC fed vs. normal diet fed mice). Secondly, there is anaccumulation of high molecular weight (HMW) aggregates (see i and ii andloss of band intensity at position iii) after DDC. Relative to controlDsiRNA, treatment with β-catenin DsiRNA led to lower HMW aggregates andincreased residual monomer at position iii, which mirrors the differencein the porphyrin levels in these samples. However, induction ofglutathione-S-transferase-mu-1 (GST mu), which is upregulated byoxidative stress, was unchanged between control and β-catenin DsiRNAafter DDC, as assessed by Coomassie staining and also validated byimmunoblotting (FIG. 19; FIG. 18 (panel B))(Hanada, S., et al. Genderdimorphic formation of mouse Mallory body formation in drug-primed mouseliver. Am. J. Pathol. 2002; 161:2019-2026). Thus, while DDC inducesprotein aggregation, the absence of β-catenin significantly alters theaggregation pattern and reduces total protein aggregates.

β-Catenin Loss Mitigates DDC-Mediated Nuclear Intermediate Filament (IF)Protein Damage.

Cytosolic (K8/K18) and nuclear (Lamin A/C, Lamin B1) IF proteins havebeen reported to be highly susceptible to porphyrin-mediatedaggregation. Since inhibition of β-catenin after DDC resulted indecreased porphyrin accumulation, we determined if IF proteinaggregation is lower in the absence of β-catenin. DDC feeding has beenreported to cause upregulation of cytosolic IF protein K8/K18. AlthoughDDC caused upregulation of both K8 and K18 in both control and β-cateninDsiRNA-treated mice, there was no statistically significant differencein the amount of aggregate formed between the two groups (FIG. 20A(panel A), FIG. 20A (panel B), upper panel, and FIG. 20C). DDC feedingalso causes a dramatic loss of the monomer form of nuclear IF proteins,and a concomitant increase in HMW aggregates. Our results showed thatinhibition of β-catenin protected against DDC-mediated loss in monomerforms of both Lamin A/C and Lamin B1 (FIG. 20A (panel C), FIG. 20A(panel D), lower panel, and FIG. 20B). Similarly, there was asignificantly higher amount of HMW Lamin A/C aggregate in DDC fedcontrol mice compared to β-catenin-inhibited mice (FIG. 20A (panels Cand D), upper panel, and FIG. 20C). Thus, loss of β-catenin does notaffect cytosolic IF protein damage, but protects against nuclear IFprotein damage.

DDC-Mediated Endoplasmic Reticulum (ER) Protein Damage and Autophagy areAttenuated by β-Catenin DsiRNA.

Porphyrins cause ER stress through a non-canonical pathway that involvesaggregation of several ER proteins (e.g., BiP, PDI, IRE1α) required forprotein quality control. Since IRE1α is essential in activating theunfolded protein response (UPR) during ER stress, we analyzed itsprotein expression before and after DDC. Under basal conditions, we sawincreased IRE1α expression in β-catenin DsiRNA treated livers (FIG.21A), which may be due to the role of β-catenin in regulating theexpression of antioxidant genes. DDC led to an increase in the formationof HMW aggregates; however, the IRE1α monomer was greater in livers ofmice treated with β-catenin DsiRNA than in control DDC livers, while theaggregate form was decreased. Mice treated with β-catenin DsiRNA alsohad markedly higher levels of both the monomer and dimer forms ofCCAAT-enhancer-binding protein homologous protein (CHOP), anothermediator of ER stress (FIG. 21B). Thus, while DDC increases theaggregation of misfolded ER proteins such as IRE1α, this effect isdecreased in the absence of β-catenin, leading to increasedfunctionality of the ER stress response.

A similar finding was observed with p62, a poly ubiquitin (Ub) bindingprotein that is upregulated after DDC. p62 increased in both control andβ-catenin DsiRNA-treated livers after DDC and was mainly contained inHMW aggregates (FIG. 21C). However, p62 monomers were also present inβ-catenin DsiRNA livers, with complete loss of monomer observed incontrols after DDC feeding, suggesting preservation of proteinfunctionality. Interestingly, although both control and β-cateninDsiRNA-treated mice show increased poly-ubiquitinated proteins afterDDC, the extent of proteasomal inhibition was similar in both groups(FIG. 21D).

Protein aggregation as a function of porphyrin accumulation also inducesan autophagic response in order to clear the misfolded proteins. Theincrease in the level of LC3B-II compared to LC3B-I in the cell is ameasure of autophagy activity in the cell. The LC3B II/I ratio wasincreased under basal conditions in mice treated with β-catenin DsiRNAcompared with controls, albeit insignificantly (FIG. 21E; FIG. 21F).However, after DDC there was no further induction of autophagy overbaseline in β-catenin DsiRNA treated mice, whereas control mice showed asignificant increase in the LC3B II/I ratio compared to baseline. In asimilar trend, control mice showed significantly higher levels of HMWLC3B aggregates than mice lacking β-catenin (FIG. 21E; FIG. 21F). Thus,DDC-induced autophagy activation is blunted in the presence of β-cateninDsiRNA, likely due to the decrease in HMW protein aggregates compared tocontrols.

In mouse models, we demonstrate a therapeutic benefit of inhibitingβ-catenin in protecting livers from porphyria-induced injury. Thisinhibition affected multiple points in the pathway, includingsuppression of CAR, ALA-D, and HO-1, and negative feedback regulation ofALA-S (FIG. 22). Although there may be negative effects of long-termβ-catenin suppression, such as counteracting oxidative stress(Nejak-Bowen, K. N., et al. Beta-catenin regulates vitamin Cbiosynthesis and cell survival in murine liver. J. Biol. Chem. 2009;284:28115-28127 and Zhang, X. F., et al. Conditional beta-catenin lossin mice promotes chemical hepatocarcinogenesis: role of oxidative stressand platelet-derived growth factor receptor alpha/phosphoinositide3-kinase signaling. Hepatology 2010; 52:954-965), and thereforesustained β-catenin inhibition may be contra-indicated, treatment cycleswith a β-catenin suppressor are believed to be both safe and efficaciousat ameliorating liver injury caused by porphyrin disorders.

Example 2

Two-month old CD-1 mice were started on a 0.1% DDC diet to induceporphyria. Three days after starting the diet, mice receivedintraperitoneal (i.p.) injections of 30 mg/kg Porcupine (PORCN)inhibitor Wnt-C59. The Wnt-C59 was delivered on a five days on, two daysoff schedule for two weeks. Mice were sacrificed 6 hours after the finaldose of Wnt-C59. Serum and livers from the control and experimentalgroups were utilized for IHC, Western blotting, and biochemical assays,as described in Example 1.

Results show that serum markers of hepatobiliary injury (ALP, ALT, AST,and total bilirubin) are significantly reduced (FIG. 23A). In addition,Wnt-C59 effectively inhibited β-catenin activity, as shown by thedecrease in glutamine synthetase (GS) in FIG. 23B and a decreasedporphyrin accumulation in the Wnt-C59 experimental group in FIG. 23C.Treatment of mice with Wnt-C59, abolishes Wnt secretion and preventsβ-catenin activation, recapitulates the findings in genetic knockout ofβ-catenin and RNAi-based inhibition of β-catenin.

Example 3

WT1/T2 mice were utilized as these mice show approximately 30% of normalhydroxymethylbilane synthase (HMBS) activity and mimic acuteintermittent porphyria (AIP) upon administration of phenobarbital, dueto the massive accumulation of porphyrin precursors. Two-month oldWT1/WT2 mice received i.p. injections of 30 mg/kg Wnt-C59. The samedosing regimen and sacrifice schedule was utilized as in Example 2.Livers from the control and experimental groups were utilized forprotein extraction and Western blotting, as described in Example 1.

Results from Western blot show that mRNA levels of ALA-S and ALA-D (thefirst two enzymes in the heme biosynthesis pathway) is significantlyreduced in Wnt-C59 WT1/WT2 mouse experimental group, as compared to thecontrol group (FIG. 24). These results were also observed with theDDC-treated mice with inhibition of β-catenin.

Example 4

An additional mechanism through which β-catenin suppression may bealleviating porphyria is through the β-catenin/GS/mTOR axis to induceautophagy. Autophagy is a potential mechanism for the clearance ofaggregated or misfolded proteins caused by porphyrin inclusions.

A group of two-month old WT1/WT2 control mice and mice injected withβ-catenin DsiRNA were fed a diet lacking DCC, as described in Example 1.In a second group, control mice and mice injected with β-catenin DsiRNAwere fed a diet containing DCC, as described in Example 1. Livers fromthe controls and β-catenin DsiRNA experimental groups were utilized forprotein extraction, SDS-Page, Western blotting, and biochemical assaysas described in Example 1.

To test the effect of Wnt-C59 on GS and p-mTOR levels, two-month oldWT1/WT2 mice were exposed to a DCC diet, as described in Example 1, andreceived i.p. injections of Wnt-C59, as described in Example 2. Liversfrom the control and experimental groups were utilized for IHC, asdescribed in Example 1.

SDS-Page was utilized to highlight the changes in the protein bandingpattern between controls and β-catenin DsiRNA experimental groups, as afunction of a DCC diet (FIG. 25A). Western blot and assays showed adecrease in LC3BI protein and an increase in LC3BII protein producedafter β-catenin DsiRNA treatment (FIG. 25B). Increased levels of LC3BIIindicated activated autophagy, as the loss of β-catenin led to the lossof p-mTOR expression. Control mice fed DCC also showed an induction ofautophagy, likely a result of increased protein aggregation. The levelof LC3B aggregates decreased in mice treated with β-catenin and DCC, ascompared to the control.

IHC of WT1/WT2 mice livers showed that β-catenin is able to regulatephospho-mTOR S2448 (p-mTOR) (FIG. 26). The expression of p-mTOR isthrough the β-catenin transcriptional regulation of GS, whichsequentially regulates the glutamine levels that can phosphorylate mTORdirectly. Control WT1/WT2 mice had both GS and p-mTOR proteins aroundthe central vein location. The expression of both GS and p-mTOR proteinsdecreased with a DCC diet, while a DCC diet and Wnt-C59 injection led tono expression of the GS and p-mTOR proteins.

Although the methods have been described in detail for the purpose ofillustration based on what is currently considered to be the mostpractical and preferred embodiments, it is to be understood that suchdetail is solely for that purpose and that the methods are not limitedto the disclosed embodiments, but on the contrary, is intended to covermodifications and equivalent arrangements that are within the spirit andscope of the appended claims. For example, it is to be understood thatthe present methods contemplate that, to the extent possible, one ormore features of any embodiment can be combined with one or morefeatures of any other embodiment.

The present invention is also directed to the following clauses:

Clause 1: A method of treating porphyria in a patient in need thereof,comprising: decreasing expression or activity of β-catenin in thepatient, thereby decreasing porphyrin production and/or accumulation, orreducing one or more symptoms of porphyria, such as liver injury in thepatient by administering to a patient an amount of a therapeutic agenteffective to decrease expression or activity of β-catenin in a patient,thereby decreasing porphyrin production and/or accumulation, or reducingone or more symptoms of porphyria, such as liver injury in a patient.

Clause 2: The method of clause 1, wherein expression of β-catenin isknocked-down in the patient.

Clause 3: The method of clause 2, wherein expression of β-catenin istransiently knocked-down in the patient.

Clause 4: The method of clause 2 or 3, wherein the expression ofβ-catenin is decreased by an RNAi reagent.

Clause 5: The method of any of clauses 2 to 4, wherein the expression ofβ-catenin is decreased by an siRNA, such as a DsiRNA reagent, specificto β-catenin.

Clause 6: The method of clause 4, wherein the RNAi reagent has thesequence of any one of SEQ ID NOS: 2-21.

Clause 7: The method of clause 2, wherein expression of β-catenin isdecreased by use of an antisense reagent, such as a nucleic acid ornucleic acid analog.

Clause 8: The method of clause 2, wherein expression of β-catenin isdecreased by use of gene editing.

Clause 9: The method of any of clauses 1 to 8, wherein activity ofβ-catenin in decreased by use of an inhibitor ofWnt/β-catenin/TCF-mediated transcription.

Clause 10: The method of clause 8, wherein the inhibitor is ICG001, oran isomer or enantiomer thereof, including racemic mixtures thereof.

Clause 11: The method of clause 8, wherein the inhibitor is PRI724.

Clause 12: The method of clause 8, wherein the inhibitor ofWnt/β-catenin/TCF-mediated transcription is a PORCN inhibitor.

Clause 13: The method of clause 12, wherein the PORCN inhibitor isLGK974, CGX1321, ETC-1922159 (ETC-159), IWP2, IWP1, IWP-O1, RXC004,IPW-3, IPW-4, GNF-6231, IWP-12, or IWP-L6.

Clause 14: The method of clause 12, wherein the PORCN inhibitor isWnt-C59.

Clause 15: The method of any of clauses 1 to 14, wherein expression oractivity of β-catenin in the liver of the patient is decreased.

Clause 16: The method of any of clauses 1 to 15, wherein the patient'sliver is targeted for decreasing expression or activity of β-catenin.

What is claimed is:
 1. A method of treating porphyria in a patient inneed thereof, comprising: decreasing expression or activity of β-cateninin the patient, thereby decreasing porphyrin production and/oraccumulation, or reducing one or more symptoms of porphyria in thepatient by administering to a patient an amount of a therapeutic agenteffective to decrease expression or activity of β-catenin in a patient,thereby decreasing porphyrin production and/or accumulation, or reducingone or more symptoms of porphyria in the patient, wherein expression oractivity of β-catenin is decreased by an RNAi reagent, an antisensereagent, or by an inhibitor of Wnt/β-catenin/TCF-mediated transcription.2. The method of claim 1, wherein expression of β-catenin is decreasedby an RNAi reagent.
 3. The method of claim 2, wherein expression ofβ-catenin is decreased by a siRNA specific to β-catenin.
 4. The methodof claim 3, wherein the RNAi reagent is a siRNA having the sequence ofany one of SEQ ID NOS: 2-21.
 5. The method of claim 1, whereinexpression of β-catenin is decreased by use of an antisense reagent. 6.The method of claim 1, wherein activity of β-catenin is decreased by useof an inhibitor of Wnt/β-catenin/TCF-mediated transcription.
 7. Themethod of claim 6, wherein the inhibitor is ICG001, or an isomer orenantiomer thereof, including racemic mixtures thereof.
 8. The method ofclaim 6, wherein the inhibitor is PRI724.
 9. The method of claim 6,wherein the inhibitor of Wnt/β-catenin/TCF-mediated transcription is aPORCN inhibitor.
 10. The method of claim 9, wherein the PORCN inhibitoris LGK974, CGX1321, ETC-1922159 (ETC-159), IWP2, IWP1, IWP-O1, RXC004,IPW-3, IPW-4, GNF-6231, IWP-12, or IWP-L6.
 11. The method of claim 9,wherein the PORCN inhibitor is Wnt-C59.
 12. The method of claim 1,wherein expression or activity of β-catenin in the liver of the patientis decreased.
 13. The method of claim 12, wherein the patient's liver istargeted for decreasing expression or activity of β-catenin.
 14. Themethod of claim 1, wherein the one or more symptoms of porphyria isliver injury.
 15. The method of claim 3, wherein the siRNA is a DsiRNAreagent.
 16. The method of claim 5, wherein the antisense reagent is anucleic acid or nucleic acid analog.